Certain strains of bacteria play an important role in the environment by acting as "Nature's biodegraders". Unactivated organic compounds, such as aliphatic hydrocarbons, phthalates, chlorinated aromatic compounds, and polycyclic aromatic hydrocarbons, are hydroxylated in the first stages of bacterial assimilation. These reactions are carried out by enzyme complexes in which the oxygen-activating component is either a P-450-type cytochrome, a flavin monooxygenase, or a nonheme iron mono- or dioxygenase. Very little information pertaining to the active-site structures and reaction mechanisms of the nonheme iron oxygenases is available. In view of this circumstance, we have targeted three representative nonheme iron oxygenases for study: Pseudomonas oleovorans omega-hydroxylase, Pseudomonas cepacia phthalate oxygenase, and Methylosinus trichosporium OB3b methane monooxygenase. During the next five years we propose to: (1) study the equilibrium interactions of these enzymes with their physiological reductases in order to determine complex stoichiometries and dissociation constants; (2) determine the pH, ionic strength, and temperature dependences of the reduction potentials of these enzymes in the presence and absence of their reductases or substrates; (3) identify the chromophoric organic species that are present in omega-hydroxylase and methane monooxygenase (component A); (4) prepare manganese- and cobalt- substituted derivatives of these enzymes; and (5) study the reactions of these enzymes with organic hydroperoxides and percarboxylic acids. Our long-term goal is to understand in detailed mechanistic terms the individual steps which constitute the catalytic cycles of these important enzymes. All three of these enzymes have additionally been shown to produce products of industrial interest in vitro; the experiments outlined in this proposal will lay a foundation for the development of electrocatalytic schemes employing them.